Application of long-chain non-coding RNA expression inhibition in esophageal cancer ferroptosis inducer sensitization

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of long-chain non-coding RNA expression inhibition in esophageal cancer ferroptosis inducer sensitization. The application first reveals dynamic change of long-chain non-coding RNA ENSG00000250658 in esophageal squamous cell carcinoma metastasis based on single-cell sequencing data, detects expression of ENSG00000250658 in esophageal squamous cell carcinoma tissue and correlation with prognosis by using LNA probe-based RNA in-situ hybridization technology, and first finds that knocking down ENSG00000250658 can increase killing effect of ferroptosis inducer on esophageal squamous cell carcinoma cells. Therefore, the application provides a new technical scheme and thought for clinical diagnosis and treatment of esophageal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of inhibiting the expression of long non-coding RNA in the sensitization of ferroptosis inducers in esophageal cancer. Background Technology

[0002] Esophageal cancer is a highly lethal malignant tumor worldwide, with esophageal squamous cell carcinoma (ESCC) being the main pathological subtype. Although the 5-year survival rate for patients with early-stage esophageal cancer can reach over 90% after treatment, most patients are already in the middle or late stages at initial diagnosis due to atypical early symptoms, resulting in local and distant metastases and a generally poor prognosis. Therefore, identifying key molecules that promote the progression and metastasis of esophageal cancer and applying them to the diagnosis, prognostic assessment, and development of therapeutic targets for esophageal cancer has significant clinical translational value.

[0003] Ferroptosis is a regulated cell death process driven by iron-dependent lipid peroxidation. Iron homeostasis is a key regulator of ferroptosis. The intracellular "unstable iron pool" maintains a dynamic balance through iron uptake, utilization, and efflux. When this balance is disrupted, ferrous ions accumulate, generating reactive oxygen species (ROS) via the Fenton reaction, triggering a chain reaction of lipid peroxidation, leading to oxidative damage to the cell membrane, and ultimately promoting ferroptosis. This process is typically accompanied by impaired antioxidant defense systems (such as decreased GPX4 expression and glutathione depletion) and activation of ACSL4, a key enzyme in lipid metabolism.

[0004] Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides. They play a regulatory role in gene transcription, translation, and post-translational modifications, and are involved in multiple processes in cancer, including abnormal proliferation, local invasion, and distant metastasis. Due to the high specificity of lncRNA expression in cancer, they have become potential biomarkers and therapeutic targets for cancer diagnosis and prognostic assessment.

[0005] ENSG00000250658 is a long non-coding RNA annotated in the Ensembl database (Ensembl release 115), located on human chromosome 4, specifically Chromosome 4: 187,226,515-187,505,647, containing 19 transcripts; the NCBI RefSeq database annotates this long non-coding RNA (Gene ID LOC339975) as a single transcript, with accession number NR_038931.1. Currently, the inventors' team has only conducted preliminary research on the function of this long non-coding RNA in esophageal squamous cell carcinoma, and found that it is highly expressed in highly invasive esophageal squamous cell carcinoma cells KYSE30. Knocking down the expression of this long non-coding RNA using small interfering RNA (siRNA), short hairpin RNA (shRNA), or antisense oligonucleotide (ASO) can inhibit the proliferation, migration, invasion, and metastasis of KYSE30 cells.

[0006] However, the expression level of ENSG00000250658 in esophageal cancer patient tissues, its association with prognosis, and whether it can serve as a potential target for ferroptosis sensitization in esophageal cancer remain unclear. Summary of the Invention

[0007] The purpose of this invention is to provide the application of inhibiting the expression of long non-coding RNA in the sensitization of ferroptosis inducers in esophageal cancer. Inhibiting or reducing the expression of long non-coding RNA can enhance the killing effect of ferroptosis inducers on esophageal cancer cells. Therefore, reagents that inhibit or reduce the expression of long non-coding RNA can be used to prepare ferroptosis sensitizers for esophageal cancer.

[0008] This invention provides the application of reagents for inhibiting or reducing the expression of long non-coding RNAs in the preparation of esophageal cancer ferroptosis sensitizers, wherein the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

[0009] Preferably, the esophageal cancer includes esophageal squamous cell carcinoma.

[0010] Preferably, the reagent for inhibiting or reducing the expression of long non-coding RNA includes shRNA and / or siRNA; the sequences of the forward and reverse oligonucleotides of the shRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the sense and antisense strands of siRNA-1 are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the nucleotide sequences of the sense and antisense strands of siRNA-2 are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0011] Preferably, the ferroptosis sensitizer enhances the killing effect of the ferroptosis inducer on esophageal cancer cells; the ferroptosis inducer includes RSL3.

[0012] The present invention also provides a drug for treating esophageal cancer, wherein the active ingredients of the drug include a reagent for inhibiting or reducing the expression of long non-coding RNA and a ferroptosis inducer; the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

[0013] Preferably, the ferroptosis inducer includes RSL3.

[0014] Preferably, the reagent for inhibiting or reducing the expression of long non-coding RNA includes shRNA and / or siRNA; the sequences of the forward and reverse oligonucleotides of the shRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the sense and antisense strands of siRNA-1 are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the nucleotide sequences of the sense and antisense strands of siRNA-2 are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0015] The present invention also provides the application of a reagent for detecting the expression of long non-coding RNA in the preparation of esophageal cancer prognostic products, wherein the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

[0016] Preferably, the reagent includes an in situ hybridization detection reagent, which includes an LNA probe for detecting the long non-coding RNA, the nucleotide sequence of which is shown in SEQ ID NO.16.

[0017] Preferably, the expression of the long non-coding RNA is upregulated in esophageal cancer samples.

[0018] Beneficial effects: This invention provides the application of reagents that inhibit or reduce the expression of long non-coding RNAs (LNAs) in the preparation of ferroptosis sensitizers for esophageal cancer, and also provides the application of reagents that detect LNA expression in the preparation of prognostic products for esophageal cancer. This invention, for the first time, reveals the dynamic changes of the long non-coding RNA ENSG00000250658 during the metastasis of esophageal squamous cell carcinoma based on single-cell sequencing data. RNA in situ hybridization technology based on LNA probes was used to detect the expression of ENSG00000250658 in esophageal squamous cell carcinoma tissues and its correlation with prognosis. Furthermore, it was discovered for the first time that knocking down ENSG00000250658 can increase the killing effect of ferroptosis inducers on esophageal squamous cell carcinoma cells. Therefore, this invention provides a new technical solution and approach for the clinical diagnosis and treatment of esophageal cancer. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 This is a graph showing the results of analyzing the dynamic changes of target long non-coding RNA in an esophageal squamous cell carcinoma lung metastasis model based on single-cell RNA sequencing data in Example 1; Figure 2 This is a graph showing the results of flow cytometry analysis in Example 2, which detected that knockdown of target long non-coding RNA inhibited the survival of esophageal squamous cell carcinoma cells in nude mouse blood via the ferroptosis pathway. Figure 3 This is a diagram showing the results of the nude mouse lung metastasis model in Example 3, which detected that knocking down the expression of the target long non-coding RNA inhibited the lung colonization of esophageal squamous cell carcinoma cells through the ferroptosis pathway. Figure 4 This is a graph showing the results of the CCK-8 assay in Example 4, which detected the effect of knockdown of target long non-coding RNA expression on the ferroptosis sensitivity of esophageal squamous cell carcinoma cells. Figure 5 This is a figure showing the results of the fluorescence probe method used in Example 5 to detect the effect of knockdown of target long non-coding RNA expression on lipid reactive oxygen species and ferrous ion levels in esophageal squamous cell carcinoma cells. Figure 6This is a figure showing the results of Western blot analysis in Example 6 on the effect of knockdown of target long non-coding RNA expression on the expression of ferroptosis-related molecules in esophageal squamous cell carcinoma cells; Figure 7 This is a figure showing the results of real-time quantitative PCR and Western blot analysis in Example 7, which detected the effect of knockdown of target long non-coding RNA expression on NCOA4 expression levels in esophageal squamous cell carcinoma cells. Figure 8 This is a graph showing the results of RNA in situ hybridization in Example 8, which detected the expression of target long non-coding RNA in the primary lesion, adjacent normal tissue, and metastatic lesion tissue of esophageal squamous cell carcinoma patients, and the relationship between its expression and patient prognosis. Detailed Implementation

[0021] This invention provides the application of reagents for inhibiting or reducing the expression of long non-coding RNAs in the preparation of esophageal cancer ferroptosis sensitizers, wherein the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

[0022] In one embodiment, the esophageal cancer includes esophageal squamous cell carcinoma. In one embodiment, the reagent for inhibiting or reducing the expression of long non-coding RNA includes a reagent for knocking down the expression of long non-coding RNA; in another embodiment, the reagent for knocking down the expression of long non-coding RNA can be shRNA and / or siRNA; the sequences of the forward and reverse oligonucleotides of the shRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the sense and antisense strands of siRNA-1 are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the nucleotide sequences of the sense and antisense strands of siRNA-2 are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0023] In one embodiment, the ferroptosis sensitizer enhances the killing effect of the ferroptosis inducer on esophageal cancer cells; in another embodiment, the ferroptosis inducer includes, but is not limited to, RSL3.

[0024] The present invention also provides a drug for treating esophageal cancer, wherein the active ingredients of the drug include a reagent for inhibiting or reducing the expression of long non-coding RNA and a ferroptosis inducer; the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

[0025] In one embodiment, the ferroptosis inducer includes RSL3. In another embodiment, the reagent for inhibiting or reducing the expression of long non-coding RNA includes a reagent for knocking down the expression of long non-coding RNA; in yet another embodiment, the reagent for knocking down the expression of long non-coding RNA can be shRNA and / or siRNA; the sequences of the forward and reverse oligonucleotides of the shRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the sense and antisense strands of siRNA-1 are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the nucleotide sequences of the sense and antisense strands of siRNA-2 are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0026] In this invention, inhibiting or reducing the expression of long non-coding RNA in esophageal cancer cells can significantly enhance the killing effect of ferroptosis inducers on esophageal cancer. Therefore, the reagents that inhibit or reduce the expression of long non-coding RNA can be combined with ferroptosis inducers as drugs for treating esophageal cancer. There is no special limitation on the combination ratio of the two, which can be set according to the needs.

[0027] The present invention also provides the application of a reagent for detecting the expression of long non-coding RNA in the preparation of esophageal cancer prognostic products, wherein the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

[0028] In one embodiment, the reagent includes an in situ hybridization (IHH) detection reagent, which includes an LNA probe for detecting the long non-coding RNA, the nucleotide sequence of which is shown in SEQ ID NO. 16. In another embodiment, the long non-coding RNA is upregulated in esophageal cancer samples; for example, esophageal squamous cell carcinoma patients with high expression of the long non-coding RNA have worse survival outcomes, suggesting that the target long non-coding RNA is a potential prognostic biomarker.

[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Dynamic changes of target long non-coding RNAs in an esophageal squamous cell carcinoma lung metastasis model were analyzed based on single-cell RNA sequencing data. In this embodiment, raw single-cell RNA sequencing data of the KYSE30 cell lung metastasis model were downloaded from the GEO (Gene Expression Omnibus) database. The data is numbered GSE249058 (the data is published in the following literature: Wong CN, ZhangY, Ru B, Wang S, Zhou H, Lin J, Lyu Y, Qin Y, Jiang P, Lee VH, Guan XY. Identification and Characterization of Metastasis-Initiating Cells in ESCC in a Multi-Timepoint Pulmonary Metastasis Mouse Model. Adv Sci (Weinh). 2024Aug;11(30):e2401590. doi: 10.1002 / advs.202401590. Epub 2024 Jun 12. PMID:38864342; PMCID: PMC11321633.) included GSM7925719 (in vitro parental cells), GSM7925720 (in vivo lung metastasis 6 hours), GSM7925721 (in vivo lung metastasis 48 hours), GSM7925722 (in vivo lung metastasis 2 months), and GSM7925723 (in vivo lung metastasis 4 months). Nineteen ENSG00000250658 transcripts from Ensemble's GTF were added to the refdata-gex-GRCh38-2024-A reference set. The reference transcriptome was reconstructed using Cell Ranger, and after alignment with the human reference genome, a gene expression matrix was obtained. After filtering low-quality cells and performing two-cell analysis, the positive proportion of the target long non-coding RNA ENSG00000250658 in each sample was analyzed. AUCell scores were also performed on positive and negative cell subsets for epithelial-mesenchymal transition (EMT) and ferroptosis suppression-related gene sets. Results are as follows: Figure 1 As shown in Figures A through C, Figure A represents the positive proportion of the target long non-coding RNA ENSG00000250658 in each sample; Figure B represents the comparison of EMT scores between positive and negative cells for the target long non-coding RNA ENSG00000250658 in all samples; and Figure C represents the comparison of ferroptosis inhibition scores between positive and negative cells for the target long non-coding RNA ENSG00000250658 in all samples. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group,p <0.001.

[0031] The results showed that the positive rate of the target long non-coding RNA ENSG00000250658 in various samples of the KYSE30 cell lung metastasis model changed significantly with metastasis progression: the positive rate was 10.5% under in vitro culture conditions; after tail vein injection, the proportion of cells colonized in lung cells increased to 40.3% at 6 hours and then surged to 73% at 48 hours; in mature lung metastases, the positive rate was 55% at 2 months and 67.1% at 4 months. Figure 1 (A). This dynamic change indicates that tumor cells positive for the target long non-coding RNA ENSG00000250658 are significantly enriched in the early metastatic stage (6-48 hours), potentially exhibiting stronger blood viability and lung colonization capacity. Consistently, the EMT and ferroptosis inhibition scores of the ENSG00000250658-positive cell subset were significantly higher than those of the negative cell subset (EMT score: Figure 1 Middle B; Ferric Morphology Inhibition Score: Figure 1 (C)

[0032] In summary, cells positive for the target long non-coding RNA ENSG00000250658 were significantly enriched in the early stage of hematogenous metastasis of esophageal squamous cell carcinoma. This enrichment was associated with ferroptosis resistance and EMT pathway activation, suggesting that the target long non-coding RNA ENSG00000250658 is a key molecule that promotes tumor cell hematogenous survival and successful colonization.

[0033] Example 2 Knocking down target long non-coding RNA inhibits the survival of esophageal squamous cell carcinoma cells in nude mouse blood via the ferroptosis pathway. 1. Establishment of mCherry and firefly luciferase-labeled target long non-coding RNA knockdown cell lines and control cell lines ① Cell Culture Esophageal squamous cell carcinoma cells KYSE30 (a gift from Professor Y. Shimada of the University of Tokyo) and HEK293T cells (ATCC) were routinely resuscitated and cultured in a 37°C, 5% CO2 incubator. KYSE30 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), while HEK293T cells were cultured in DMEM medium containing 10% FBS. 1% penicillin / streptomycin was added to both media.

[0034] ② Virus packaging and establishment of mCherry and firefly luciferase double-labeled knockdown cell lines The pCDH-MCS-EF1-Hygro-mCherry plasmid carrying the mCherry reporter gene was transfected using Neofect™ DNA transfection reagent (Beijing Codey Co., Ltd.). (This plasmid was published in the following literature: Sun Y, Chen D, Sun S, Ren M, Zhou L, Chen C, Zhao J, Wei H, Zhao Q, Qi Y, Zhang J, Zhang G, Liu H, Yang Q, Liu Q, Wang Y, Zhang W. RBMS1 Coordinates with the m 6 A Reader YTHDF1 to Promote NSCLC Metastasis through Stimulating S100P Translation. Adv Sci(Weinh). 2024 Apr;11(15):e2307122. doi: 10.1002 / advs.202307122. Epub 2024 Feb11. PMID: 38342601; PMCID: PMC11022699) was co-transfected with lentiviral packaging plasmids (psPAX2 and pMD2.G) into HEK293T cells. After 48-72 hours, the cell supernatant containing lentiviral particles was collected, filtered through a 0.45 μm filter, and then used to infect KYSE30 cells for 24 hours with the infection-promoting agent polybrene. Subsequently, cells were continuously screened with hygromycin for 5 days, and the infection efficiency was observed and verified by fluorescence microscopy.

[0035] Next, shRNA lentiviruses labeled with firefly luciferase targeting the target long non-coding RNA and control lentiviruses (shCtrl) were used to infect the aforementioned mCherry-labeled KYSE30 cells. After selection with puromycin, KYSE30 cells with target long non-coding RNA double-labeled by mCherry and firefly luciferase and control cells were obtained. Uninfected mCherry-labeled KYSE30 cells were used as a negative control. After 1-2 days of puromycin selection, all negative control cells died, indicating that the selection conditions were effective.

[0036] The aforementioned shRNA lentiviruses targeting long non-coding RNAs and control lentiviruses were packaged by Shanghai Jikai Gene Technology Co., Ltd. The specific packaging steps are briefly described below: The two synthesized oligonucleotides (SEQ ID NO:1 and SEQ ID NO:2) were annealed to form double-stranded DNA with sticky ends, which was then constructed into the GV344 vector to obtain the shRNA recombinant vector. Subsequently, the shRNA recombinant vector and packaging helper plasmids (Helper 1.0 and Helper 2.0) were co-transfected into HEK293T cells to obtain shRNA lentiviruses targeting the target long non-coding RNA.

[0037] The information for the two oligonucleotide sequences is as follows: The forward oligonucleotide of shRNA is: 5'-CCGGGACTTCCACGAACTGAACCAGTGTTCTCGAGAACACTGGTTCAGTTCGTGGAAGTCTTTTTG-3' (SEQ ID NO:1); the reverse oligonucleotide of shRNA is: 5'-AATTCAAAAAGACTTCCACGAACTGAACCAGTGTTCTCGAGAACACTGGTTCAGTTCGTGGAAGTC-3' (SEQ ID NO:2).

[0038] The control lentivirus is constructed by packaging a nonsense sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO:3) into a GV344 vector.

[0039] ③ Verification of cell line knockdown efficiency Total RNA was extracted from the selected surviving cells using RNAiso Plus (TaKaRa) reagent, and the RNA was reverse transcribed into cDNA template using the HiScript IV RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd.). Then, real-time quantitative PCR was performed using the ChamQ Universal SYBRqPCR Master Mix reagent (Nanjing Novizan Biotechnology Co., Ltd.).

[0040] The primer information for real-time quantitative PCR is as follows: The upstream sequence of the primer for specifically amplifying the target long non-coding RNA is: 5'-ACGGAAAAGACTTCCACGAAC-3' (SEQ ID NO:4); the downstream sequence of the primer for specifically amplifying the target long non-coding RNA is: 5'-TCACTTGGCACCCCGACA-3' (SEQ ID NO:5); the upstream sequence of the GAPDH primer is: 5'-ACAACTTTGGTATCGTGGAAGG-3' (SEQ ID NO:6); the downstream sequence of the GAPDH primer is: 5'-GCCATCACGCCACAGTTTC-3' (SEQ ID NO:7).

[0041] Data collection and processing: Using GAPDH as an internal reference gene, 2 -△△CT The expression levels of target long non-coding RNAs were analyzed using a method with three parallel wells per group. Results are presented as mean ± standard deviation, and statistical analysis was performed using the unpaired Student's t-test. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group, p <0.001. The result is as follows. Figure 2 As shown in Figure A, compared with cells infected with control lentivirus, the expression level of the target long non-coding RNA was significantly reduced in cells infected with shRNA lentivirus, indicating that the knockdown cell line was successfully established.

[0042] 2. Detection of circulating tumor cells in the blood of nude mice KYSE30 cells and control cells (infected with control lentivirus) were knocked down with the target long non-coding RNA double-labeled by mCherry and firefly luciferase. The cells were pretreated with DMSO and the ferroptosis inhibitor Liproxstatin-1 (Lip-1; 1 μM) for 48 hours, respectively. Cells were then routinely digested, counted, and resuspended in PBS for later use. Four- to six-week-old male BALB / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into a negative control group and a target long non-coding RNA knockdown group, with six mice in each group. Each mouse was injected intravenously with 100 μL of cell suspension (containing 1×10⁻⁶ cells / mL). 6(cells). 24 hours after injection, blood from nude mice was collected in EDTA anticoagulant tubes, and 10 times the volume of erythrocyte lysis buffer was added to treat the cells for 5 minutes. The cells were centrifuged at 300 ×g at room temperature for 10 minutes. The supernatant was discarded, and the cell pellet free of visible erythrocyte contamination was collected. FITC-labeled anti-mouse CD45 antibody (Biolegend) was added, and the cells were incubated at 4°C for 30 minutes. After washing twice with PBS, the cell pellet was resuspended in PBS, and mCherry+ CD45 was detected by flow cytometry. - Cell ratio. The animal experiments in this embodiment have been approved by the Laboratory Animal Welfare and Ethics Committee of Dalian Medical University, and all operations strictly follow relevant ethical guidelines.

[0043] The results are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group, p <0.05; This indicates that compared with the control group, p <0.01; # indicates no statistically significant difference compared to the control group. Results are as follows: Figure 2 As shown in Figure B, knocking down the expression of the target long non-coding RNA significantly reduced the survival ability of KYSE30 cells in the blood; however, cell survival in the blood was significantly restored after Lip-1 pretreatment.

[0044] Example 3 Knocking down target long non-coding RNA inhibits the colonization of esophageal squamous cell carcinoma cells in nude mouse lung tissue via the ferroptosis pathway. KYSE30 cells and control cells (infected with control lentivirus) were knocked down with the target long non-coding RNA double-labeled by mCherry and firefly luciferase, as described in Example 2. The cells were pretreated with DMSO and the ferroptosis inhibitor Lip-1 (1 μM) for 48 hours, respectively. Cells were then routinely digested, counted, and resuspended in PBS for later use. Four- to six-week-old male BALB / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into a negative control group and a target long non-coding RNA knockdown group, with five mice in each group. Each mouse received a tail vein injection of 100 μL of cell suspension (containing 1×10⁻⁶ cells). 6 (cells). Twenty-four hours after injection, 150 mg / kg of D-fluorescein potassium salt was injected intraperitoneally into the nude mice according to their body weight. The mice were euthanized 15 minutes later, and lung tissue was removed. Bioluminescent signals in the mouse lungs were detected using a small animal in vivo imaging system. The animal experiments in this embodiment have been approved by the Experimental Animal Welfare and Ethics Committee of Dalian Medical University, and all procedures strictly adhered to relevant ethical guidelines.

[0045] Results are presented as mean ± standard deviation. Statistical analysis was performed using the unpaired Student's t-test. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group, p <0.01. The result is as follows: Figure 3 As shown, knocking down the expression of the target long non-coding RNA significantly reduced the colonization ability of KYSE30 cells in lung tissue; however, after Lip-1 pretreatment, the colonization signal of cells in lung tissue was significantly restored.

[0046] Example 4 Knocking down target long non-coding RNA enhances the sensitivity of esophageal squamous cell carcinoma cells to ferroptosis inducers. 1. siRNA-mediated knockdown of target long non-coding RNA expression This embodiment utilizes two siRNAs (siRNA-1 and siRNA-2) to knock down the expression of the target long non-coding RNA. The siRNAs used were Stealth RNAi siRNAs purchased from Invitrogen, and corresponding random nucleotides were synthesized as negative control siRNAs (siCtrl). The sense strand of siRNA-1 is: 5'-CCCAAAUUGUGGGAUUAAAUGGCAU-3' (SEQ ID NO:8); the antisense strand of siRNA-1 is: 5'-AUGCCAUUUAAUCCCACAAUUUGGG-3' (SEQ ID NO:9); the sense strand of siRNA-2 is: 5'-GACUUCCACGAACUGAACCAGUGUU-3' (SEQ ID NO:10); the antisense strand of siRNA-2 is: 5'-AACACUGGUUCAGUUCGUGGAAGUC-3' (SEQ ID NO:11).

[0047] Log-phase KYSE30 cells were transfected with siRNA-1 and siRNA-2 (targeting long non-coding RNAs) and a negative control siRNA using Invitrogen's Lipofectamine 2000 transfection reagent. After 48 hours, total RNA was extracted from the transfected cells and reverse transcribed as described in Example 2. The expression level of the target long non-coding RNA was detected by real-time quantitative PCR. Results are presented as mean ± standard deviation and statistical analysis was performed using one-way ANOVA. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group,p <0.001. The result is as follows. Figure 4 As shown in Figure A, compared with the control group cells transfected with negative control siRNA, the expression level of the target long non-coding RNA was significantly reduced in cells transfected with siRNA-1 and siRNA-2.

[0048] 2. CCK-8 assay to detect the effect of knockdown of target long non-coding RNA on sensitivity to ferroptosis inducers To evaluate the sensitizing effect of knocking down the target long non-coding RNA on the ferroptosis inducer RSL3, this example uses the CCK-8 reagent to detect the growth viability of KYSE30 cells and control cells after treatment with the ferroptosis inducer alone and in combination with various ferroptosis inhibitors. The specific procedures are as follows: ① Cell transfection: Following the above method, siRNA-1 and siRNA-2 targeting the target long non-coding RNA and negative control siRNA were transfected into KYSE30 cells.

[0049] ② Cell seeding: 24 hours after transfection, the cells were routinely digested and seeded into 96-well plates at 5000 cells per well (resuspended in 100 μL of RPMI-1640 medium containing 10% FBS), with 4 parallel wells per group; a blank control group was set up at the same time, which only added an equal amount of medium and did not seed cells.

[0050] ③ Drug treatment: 96-well plates were incubated in a 37°C, 5% CO2 incubator. After 12 hours, DMSO and different concentrations of RSL3 (0.1 μM, 0.15 μM, 0.2 μM) were added to treat the cells alone for 24 hours, or RSL3 (0.2 μM) was combined with the ferroptosis inhibitor deferoxamine (DFO; 20 μM), the apoptosis inhibitor Z-VAD-FMK (40 μM), the necrosis inhibitor GSK872 (5 μM), the autophagy inhibitor 3-methyladenine (3-MA; 5 mM), or chloroquine (CQ; 20 μM) to treat the cells for 24 hours.

[0051] ④ Detect absorbance: Replace the medium with CCK-8 reagent (the ratio of CCK-8 reagent to medium is 1:10) and continue incubation for 1 hour. Then, use an ELISA reader to measure the absorbance (OD value) at a wavelength of 450 nm.

[0052] The results are presented as mean ± standard deviation, and statistical analysis was performed using two-way ANOVA. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group, p <0.001. The result is as follows. Figure 4 Figures B and C show the results of KYSE30 cells' sensitivity to RSL3 drug after knocking down the expression of the target long non-coding RNA using the CCK-8 assay; and the results of various death inhibitors' effects on KYSE30 cells' sensitivity to RSL3 drug after knocking down the expression of the target long non-coding RNA using the CCK-8 assay.

[0053] Depend on Figure 4 As shown in Figure B, compared with the control group cells transfected with negative control siRNA, the cell viability of the target long non-coding RNA knockdown group was significantly reduced under RSL3 treatment, indicating that knockdown of the target long non-coding RNA can increase the sensitivity of esophageal squamous cell carcinoma cells to ferroptosis inducers. Figure 4 As shown in Figure C, the ferroptosis inhibitor DFO reversed the decrease in cell viability caused by RSL3 and knockdown of target long non-coding RNA, while apoptosis and necrosis inhibitors had almost no effect on restoring cell viability, although autophagy inhibitors could restore cell viability to some extent. These results indicate that ferroptosis is the main cause of cell death due to RSL3 knockdown of target long non-coding RNA, and autophagy may also be involved.

[0054] Example 5 Knocking down the target long non-coding RNA increased lipid reactive oxygen species and ferrous ion levels in esophageal squamous cell carcinoma cells. Intracellular lipid reactive oxygen species (ROS) levels and ferrous ion levels are two important biochemical indicators of ferroptosis. Therefore, this study investigated the effects of knocking down target long non-coding RNA on lipid ROS and ferrous ions in esophageal squamous cell carcinoma cells.

[0055] Lipid reactive oxygen species (ROS) detection: Log-phase KYSE30 cells were transfected with siRNA-1 and siRNA-2 (targeting long non-coding RNAs) and a negative control siRNA using Invitrogen's Lipofectamine 2000 transfection reagent. After 24 hours, cells were treated with DMSO or RSL3 (0.2 μM) for 12 hours. Cells were then washed twice with PBS, and incubated for 20 minutes at 37°C in a 5% CO2 cell culture incubator with a final concentration of 2 μM of BODIPY 581 / 591 C11 probe (Shanghai Beyotime Biotechnology Co., Ltd.). After staining, cells were digested, washed twice with PBS, resuspended in PBS, and fluorescence intensity was immediately detected by flow cytometry.

[0056] Ferrous ion detection: using ferrous ions (Fe2+) from cells. 2+The level of ferrous ions in cells was determined using a fluorescence detection kit (Dalian Meilun Biotechnology Co., Ltd.). Logarithmically growing KYSE30 cells were transfected with siRNA-1 and siRNA-2 (targeting long non-coding RNAs) and a negative control siRNA using Invitrogen's Lipofectamine 2000 transfection reagent. After 24 hours, cells were treated with DMSO or RSL3 (0.2 μM) for 12 hours. Cells were then washed twice with cell staining buffer, and incubated with a final concentration of 2 μM of the fluorescent probe at 37°C and 5% CO2 for 30 minutes. After staining, images were captured using a fluorescence microscope; the intensity of the orange-red fluorescence directly reflects the level of ferrous ions in the cells.

[0057] The results are as follows Figure 5 As shown in Figures A and B, A represents the results of detecting lipid reactive oxygen species in KYSE30 cells and control cells using the BODIPY 581 / 591 C11 probe to knock down the target long non-coding RNA, while B represents the results using cellular ferrous ions (Fe2+). 2+ The results of detecting ferrous ions in KYSE30 cells and control cells by using a fluorescence detection kit to detect the knockdown of ferrous ions by the target long non-coding RNA.

[0058] from Figure 5 As shown in Figures A and B, under RSL3 treatment, compared with the control group cells transfected with negative control siRNA, the levels of intracellular lipid reactive oxygen species and ferrous ions were increased in the target long non-coding RNA knockdown group, further indicating that knockdown of the target long non-coding RNA can enhance the ferroptosis susceptibility of esophageal squamous cell carcinoma cells.

[0059] Example 6 Knocking down target long non-coding RNA alters the expression of ferroptosis-related molecules. Log-phase KYSE30 cells were transfected with siRNA-1 and siRNA-2 (targeting long non-coding RNAs) and a negative control siRNA using Invitrogen's Lipofectamine 2000 transfection reagent. After 48 hours, cells were collected and lysed with RIPA lysis buffer (containing protease inhibitors), followed by centrifugation at 12,000 rpm for 25 minutes at 4°C. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit. Equal amounts of protein samples from each group were separated by SDS-PAGE gel electrophoresis, transferred to PVDF membranes, and blocked with 5% skim milk blocking buffer at room temperature for 1 hour. The blocked PVDF membranes were incubated overnight at 4°C with primary antibodies against GPX4 (Proteintech, 1:1000), ACSL4 (Proteintech, 1:1000), FTH1 (Proteintech, 1:2000), and GAPDH (Proteintech, 1:5000), respectively. After washing three times with TBST buffer, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. After secondary antibody incubation, the membranes were washed three more times with TBST buffer. Finally, protein bands were developed using an enhanced chemiluminescence reagent.

[0060] Western blot results are as follows Figure 6 As shown, compared with control cells transfected with negative control siRNA, the expression of antioxidant proteins GPX4 and ferritin heavy chain FTH1 was decreased in cells of the target long non-coding RNA knockdown group, while the expression of lipid metabolism enzyme ACSL4 was upregulated. These results indicate that knockdown of target long non-coding RNA can inhibit the ferroptosis antagonistic pathway and enhance the ferroptosis-promoting pathway.

[0061] In summary, all the above results demonstrate that knocking down the target long non-coding RNA can induce ferroptosis in esophageal squamous cell carcinoma cells through biochemical and molecular processes. These characteristics largely depend on the intervention of ferroptosis inducers, suggesting that the ferroptosis resistance of the target long non-coding RNA is fully manifested under stress, and that peripheral blood in the body is under high levels of oxidative stress. Therefore, the cellular results and animal experiments corroborate each other, jointly explaining why the target long non-coding RNA can resist ferroptosis and play a protective role against tumor cells during hematogenous metastasis.

[0062] Example 7 Knocking down the target long non-coding RNA promotes NCOA4 expression levels. Ferritinophagy is a selective autophagy process that specifically degrades ferritin, an intracellular iron storage protein, leading to the accumulation of free iron and ultimately ferroptosis. Nuclear receptor coactivator 4 (NCOA4) is a key regulator of ferritinophagy, transporting ferritin to lysosomes via autophagosomes. As shown in Figure C of Example 4, in addition to ferroptosis inhibitors, the autophagy inhibitors 3-MA and CQ can also reverse RSL3-induced target long non-coding RNA knockdown in KYSE30 cells to some extent. Therefore, this example investigated the regulatory role of target long non-coding RNA on NCOA4 expression.

[0063] Log-phase KYSE30 cells were transfected with siRNA-1 and siRNA-2 (targeting long non-coding RNA) and a negative control siRNA using Invitrogen's Lipofectamine 2000 transfection reagent. After 48 hours, total RNA was extracted from the transfected cells and reverse transcribed as described in Example 2. The mRNA expression level of NCOA4 was detected by real-time quantitative PCR. Alternatively, proteins were extracted from the transfected cells as described in Example 6, and the protein expression level of NCOA4 was detected by Western blot.

[0064] The primer information for real-time quantitative PCR is as follows: The upstream sequence of the NCOA4 primer is 5'-GAGGTGTAGTGATGCACGGAG-3' (SEQ ID NO:12); the downstream sequence of the NCOA4 primer is 5'-GACGGCTTATGCAACTGTGAA-3' (SEQ ID NO:13); the upstream sequence of the ACTB primer is 5'-CTCCATCCTGGCCTCGCTGT-3' (SEQ ID NO:14); the downstream sequence of the ACTB primer is 5'-GCTGTCACCTTCACCGTTCC-3' (SEQ ID NO:15).

[0065] Real-time quantitative PCR results are presented as mean ± standard deviation. ACTB was used as an internal reference gene. One-way ANOVA was used for statistical analysis. p <0.05 indicates a statistically significant difference. This indicates that compared with the control group, p <0.05; This indicates that compared with the control group, p <0.01. The result is as follows: Figure 7As shown in Figures A and B, Figure A represents the results of real-time quantitative PCR detection of the effect of knockdown of target long non-coding RNA expression on the mRNA expression level of NCOA4 in KYSE30 cells, and Figure B represents the results of Western blot detection of the effect of knockdown of target long non-coding RNA expression on the protein expression level of NCOA4 in KYSE30 cells.

[0066] Depend on Figure 7 As shown in Figures A and B, compared with the control group cells transfected with negative control siRNA, the mRNA and protein expression levels of NCOA4 in the target long non-coding RNA knockdown group were both upregulated, suggesting that knockdown of the target long non-coding RNA can promote ferritin autophagy in esophageal squamous cell carcinoma cells.

[0067] Example 8 RNA in situ hybridization was used to detect the expression of target long non-coding RNAs in esophageal squamous cell carcinoma tissues and to analyze their prognostic correlation. The two tissue microarrays used in RNA in situ hybridization were both purchased from Shanghai Chipchao Biotechnology Co., Ltd. The first microarray contained cancerous tissue and paired adjacent normal tissue from esophageal squamous cell carcinoma patients, as well as patient prognostic information. The second microarray contained the primary lesion, lymph node metastases, and distant metastases from esophageal squamous cell carcinoma patients. The probes used were 5' / 3' didigoxigenin (DIG) labeled locked nucleic acid (LNA) probes with the sequence 5'-UUCCAAUCACCUCGACGCU-3' (SEQ ID NO:16), purchased from Exqion.

[0068] RNA in situ hybridization: After dewaxing with xylene and rehydrating with gradient ethanol, tissue sections were incubated at 37°C for 2 minutes with 5 μg / mL proteinase K solution to expose the target RNA. After dehydration, LNA probe (200 nM) diluted with hybridization buffer was added and hybridized at 50°C for 1 hour. The sections were then washed with gradient SSC buffer to remove non-specific binding. Blocking buffer was added and the sections were blocked at room temperature for 15 minutes. Anti-digoxigenin antibody diluted 1:800 was added and the sections were incubated at 4°C overnight. After washing with PBST buffer, BCIP / NBT substrate chromogenic solution was added and the sections were developed in the dark at 30°C, followed by PBST washing. The sections were stained with nuclear solid red for 10 seconds, rinsed immediately, air-dried at room temperature, and mounted with neutral resin.

[0069] Results Interpretation: Staining intensity (0: no staining; 1: weak staining; 2: moderate staining; 3: strong staining) and the percentage of positively stained cells (0: <5%; 1: 5-25%; 2: 26-50%; 3: 51-75%; 4: >75%) were scored separately, and the product of the two scores was used as the final composite score. The paired Student's t-test was used. Figure 8(A) and One-wayANOVA ( Figure 8 Statistical analysis was conducted on (D). p <0.05 indicates a statistically significant difference. This indicates that compared with the control group, p <0.05; This indicates that compared with the control group, p <0.001; # indicates no statistically significant difference compared to the control group. Results are as follows: Figure 8 As shown in A and D, A is a visual diagram (left) and a statistical diagram (right) of the expression of the target long non-coding RNA in the first microarray (containing cancerous tissue and adjacent tissue of esophageal squamous cell carcinoma patients), and D is a statistical diagram of the expression of the target long non-coding RNA in the second microarray (containing primary lesions, lymph node metastases and distant metastases of esophageal squamous cell carcinoma patients).

[0070] Figure 8 As shown in Figure A, the target long non-coding RNA scored significantly higher in esophageal squamous cell carcinoma tissue than in adjacent normal tissue, indicating that the target long non-coding RNA was upregulated in esophageal squamous cell carcinoma tissue. Figure 8 The results showed that, compared with primary tumors without distant metastases, the scores of target long non-coding RNAs were significantly increased in both primary tumors with distant metastases and distant metastatic lesions, suggesting that target long non-coding RNAs are closely related to metastasis.

[0071] Subsequently, receiver operating characteristic (ROC) curve analysis was used to analyze the diagnostic efficacy of the target long non-coding RNA in the first chip. The results are as follows: Figure 8 As shown in Figure B, the area under the ROC curve (AUC) was 0.7999 (95% CI: 0.7181-0.8817), the sensitivity was 64.29%, and the specificity was 82.14%, indicating that the target long non-coding RNA has considerable diagnostic value for esophageal squamous cell carcinoma.

[0072] Furthermore, Kaplan-Meier survival curve analysis was used to analyze the association between the expression of target long non-coding RNA in the first microarray and the survival of patients with esophageal squamous cell carcinoma, and the Log-rank test was used to compare survival differences between groups. p <0.05 was considered statistically significant. Results are as follows: Figure 8 As shown in Figure C, esophageal squamous cell carcinoma patients with high expression of target long non-coding RNA had worse survival outcomes, suggesting that target long non-coding RNA is a potential prognostic biomarker.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents that inhibit or reduce the expression of long non-coding RNAs in the preparation of esophageal cancer ferroptosis sensitizers, wherein the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; and the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

2. The application according to claim 1, characterized in that, The esophageal cancer mentioned includes esophageal squamous cell carcinoma.

3. The application according to claim 2, characterized in that, The reagents for inhibiting or reducing the expression of long non-coding RNAs include shRNA and / or siRNA; the sequences of the forward and reverse oligonucleotides of the shRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the sense and antisense strands of siRNA-1 are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the nucleotide sequences of the sense and antisense strands of siRNA-2 are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

4. The application according to any one of claims 1 to 3, characterized in that, The ferroptosis sensitizer enhances the killing effect of the ferroptosis inducer on esophageal cancer cells; the ferroptosis inducer includes RSL3.

5. A drug for treating esophageal cancer, characterized in that, The active ingredients of the drug include reagents that inhibit or reduce the expression of long non-coding RNAs and ferroptosis inducers; the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

6. The drug according to claim 5, characterized in that, The ferroptosis inducer includes RSL3.

7. The drug according to claim 5 or 6, characterized in that, The reagents for inhibiting or reducing the expression of long non-coding RNAs include shRNA and / or siRNA; the sequences of the forward and reverse oligonucleotides of the shRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the siRNA includes siRNA-1 or siRNA-2; the nucleotide sequences of the sense and antisense strands of siRNA-1 are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively; the nucleotide sequences of the sense and antisense strands of siRNA-2 are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

8. Application of reagents for detecting long non-coding RNA expression in the preparation of esophageal cancer prognostic products, wherein the accession number of the long non-coding RNA locus in the Ensembl database is ENSG00000250658; the transcripts of the long non-coding RNA include 19 transcripts annotated in the Ensembl database.

9. The application according to claim 8, characterized in that, The reagents include an in situ hybridization detection reagent, which includes an LNA probe for detecting the long non-coding RNA, the nucleotide sequence of which is shown in SEQ ID NO.

16.

10. The application according to claim 8, characterized in that, The expression of the long non-coding RNA was upregulated in esophageal cancer samples.

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